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| United States Patent |
6,198,107
|
|
Seville
|
March 6, 2001
|
Fluorometric detection using visible light
Abstract
Systems, devices and methods are provided for viewing a pattern of
fluorophors capable of fluorescing when exposed to visible light, e.g.,
fluorescently stained DNA, protein or other biological material. The
system includes a light source emitting light in the visible spectrum,
such as a fluorescent lamp used in domestic lighting, a first optical
filter capable of transmitting light from the source at wavelengths
capable of exciting the fluorophors and of absorbing light of other
wavelengths, and a second optical filter capable of blocking substantially
all the light from the source not blocked by the first filter, so that the
only light reaching the viewer is light produced by fluorescence of the
fluorophors.
| Inventors:
|
Seville; Mark (Denver, CO)
|
| Assignee:
|
Clare Chemical Research, Inc. (Denver, CO)
|
| Appl. No.:
|
036034 |
| Filed:
|
March 6, 1998 |
| Current U.S. Class: |
250/458.1; 356/417 |
| Intern'l Class: |
G01N 021/64 |
| Field of Search: |
250/459.1,458.1
|
References Cited
U.S. Patent Documents
| 3802102 | Apr., 1974 | Licciardi.
| |
| 4071883 | Jan., 1978 | Dennis.
| |
| 4117338 | Sep., 1978 | Adrion et al.
| |
| 5108179 | Apr., 1992 | Myers.
| |
| 5327195 | Jul., 1994 | Ehr.
| |
| 5347342 | Sep., 1994 | Ehr.
| |
| 5387801 | Feb., 1995 | Gonzalez et al.
| |
| 5543018 | Aug., 1996 | Stevens et al.
| |
| 5736744 | Apr., 1998 | Johannsen et al. | 250/505.
|
| Foreign Patent Documents |
| 603783 A1 | Jun., 1994 | EP.
| |
Other References
Brunk, C.F. and Simpson, L., "Comparison of various ultraviolet sources for
fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide
gels," (1977) Analytical Biochemistry 82:455-462.
Grundemann, D. and Schomig, E., "Protection of DNA During Preparative
Agarose Gel Electrophoresis Against Damage Induced by Ultraviolet Light,"
Bio Techniques (Nov. 1996) 21:898-903.
Haughland, R.P., "Hand Book of Fluorescent Probes and Research Chemicals,"
(1996) 6.sup.th Edition, Michelle T.Z. Spence, Ed., Molecular Probes Inc.,
Eugene OR, pp. 13-18, 25-28 and 29-35.
Sharp, P.A. et al., "Detection of two restriction endonucleases activities
in Haemophilus parainfluenzae using analytical agarose-ethidium bromide
electrophoresis," (1973) Biochemistry 12:3055-3063.
Menzel, E.R., "An introduction to lasers, forensic lights and fluorescent
fingerprint detection techniques," (1991), Lightning Powder Company, Inc.,
Salem OR.
|
Primary Examiner: Hannaher; Constantine
Attorney, Agent or Firm: Greenlee, Winner and Sullivan, P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Ser. No. 60/040,124 filed Mar.
7, 1997, incorporated herein by reference to the extent not inconsistent
herewith.
Claims
What is claimed is:
1. A visible light transillurninator system for viewing a pattern of
fluorescence emitted by fluorophors capable of being excited by blue
exciting light, and of producing light of an emitted type, wherein said
emitted type is of a wavelength different from said blue exciting light,
said system comprising:
a) a blue non-laser light source, with maximal output in the blue
wavelength region housed within said transilluminator;
b) a blue filter placed between said light source and said fluorophors and
serving as a support for a material containing said fluorophors, which is
capable of transmitting said exciting light from said light source and of
preventing transmission of light from said light source in the wavelength
range of said emitted type by said fluorophors; and
c) an amber filter placed between said fluorophors and a light detector
which is capable of transmitting light of said emitted type from said
fluorophors and of preventing transmission of said exciting light from
said light source; said system being constructed and arranged such that
patterns of fluorescence emitted by said fluorophors are viewable.
2. The transilluminator system of claim 1 wherein said amber filter is
fitted over the top of said transilluminator.
3. The transilluminator system of claim 1 wherein said amber filter is
attached to a light detector.
4. The transilluminator system of claim 1 wherein said amber filter is
incorporated into an eyeglass.
5. A hand-held visible light system for viewing a pattern of fluorescence
emitted by fluoropbors capable of being excited by blue exciting light,
and of producing light of an emitted type wherein said emitted type is of
a wavelength different from said blue exciting light, said system
comprising:
a) a hand-held non-laser blue light source, with maximum output in the blue
wavelength region comprising a blue light and a first blue filter, said
first filter being capable of transmitting said exciting light from said
light source and of preventing transmission of light from said light
source in the wavelength range of light of said emitted type by said
fluorophors; and
b) an amber filter between said fluorophors and a human eye and/or other
light detector which is capable of transmitting light of said emitted type
from said fluorophors and of preventing transmission of said exciting
light from said light source;
said system being constructed and arranged such that patterns of
fluorescence emitted by said fluorophors are viewable.
6. The hand-held visible light system of claim 5 wherein said amber filter
is attached to said light detector.
7. The hand-held visible light system of claim 5 wherein said amber filter
is incorporated into an eyeglass.
8. A visible light system for viewing patterns of fluorescence emitted by
fluorophors capable of emitting light of an emitted light type when
excited by light of an excitation type different from said emitted type,
wherein at least a detectable portion of said emitted type is visible
light; said system comprising:
a) a light source of 40 W or less having the intensity of a household light
or less, capable of producing visible light of said excitation type for
said fluorophors;
b) a first optical filter placed between said light source and said
fluorophors which is capable of transmitting light from said light source
of said excitation type and of preventing transmission of light from said
light source of said emitted type; and
c) a second optical filter placed between said fluorophors and a light
detector to form a viewable image of the pattern of fluorophors wherein
said second filter is capable of transmitting light of said emitted type
and of preventing transmission of light of said excitation type.
9. A method of viewing a pattern of fluorescence emitted by fluorophors
capable of being excited by blue exciting light and of producing light of
an emitted type, wherein said emitted type is of a wavelength different
from said blue exciting light, said method comprising:
a) passing light from a blue non-laser light source, with maximal output in
the blue wavelength region and housed within a transilluminator, through a
first blue filter, serving as a support for a material containing said
fluorophors, capable of transmitting said exciting light from said light
source and capable of preventing transmission of light from said light
source in the wavelength range of light of said emitted type, on to said
material containing said fluorophors, whereby said fluorophors emit light
of said emitted type;
b) passing said light emitted by said fluorophors through a second amber
filter which is capable of transmitting light of said emitted type and of
preventing transmission of said exciting light from said light source,
whereby an image of said pattern of fluorescence is formed; and
c) viewing said image with a human eye and/or other light detector.
10. The method of claim 9 wherein said fluorophors are comprised in
fluorescently stained and/or labeled DNA.
11. The method of claim 9 wherein said fluorophors comprise at least one
fluorescently stained and/or labeled protein.
12. The method of claim 9 wherein said fluorophors are comprised in a gel.
13. The method of claim 9 wherein said fluorophors are comprised in a
living organism.
14. The method of claim 9 wherein said fluorophors are viewed in an array
of test tubes.
Description
BACKGROUND OF THE INVENTION
The separation of DNA fragments by polyacrylamide or agarose gel
electrophoresis is a well-established and widely used tool in molecular
biology (Sharp, P. A. et al., "Detection of two restriction endonucleases
activities in Haemophilus parainfluenzae using analytical agarose-ethidium
bromide electrophoresis," (1973) Biochemistry 12:3055). The standard
technique for viewing the positions of the separated fragments in a gel
involves the use of an ultra-violet (UV) transilluminator (Brunk, C. F.
and Simpson, L., "Comparison of various ultraviolet sources forfluorescent
detection of ethidium bromide-DNA complexes in polyacrylamide gels,"
(1977) Analytical Biochemistry 82:455). This procedure involves first
staining the gel with a fluorescent dye such as ethidium bromide or
SYBR.RTM. Green I. The DNA fragments, which bind the dye, are then
visualized by placing the gel on a light-box equipped with a UV
light-source. Typically the UV source, in combination with a built-in
filter, provides light with an excitation maximum of around 254, 300 or
360 nm. The UV light causes the DNA-bound dye to fluoresce in the red
(ethidium bromide) or green (SYBR Green I) regions of the visible light
spectrum. The colored fluorescence allows visualization and localization
of the DNA fragments in the gel. The visualization of DNA in a gel is used
either to assess the success of a gene cloning reaction as judged by the
size and number of DNA fragments present, or to identify a particular
sized fragment which can be cut out from the gel and used in further
reaction steps.
Transilluminators used in the art to visualize fluorophors are described in
a number of patents, including U.S. Pat. Nos. 5,347,342, 5,387,801,
5,327,195, 4,657,655, and 4,071,883. Clinical examination of skin
anomalies causing fluorescence have been described in U.S. Pat. No.
5,363,854 using visible light images as a control.
The use of UV light for viewing molecules in gels has two major
disadvantages: (1) It is dangerous. The eyes are very sensitive to UV
light and it is an absolute necessity that the viewer wear eye-protection,
even for brief viewing periods, to prevent the possibility of serious
damage. More prolonged exposure to UV light results in damage to the skin
tissues (sunburn) and care must be taken to minimize skin exposure by
wearing gloves, long-sleeved jackets and a full-face mask. (2) DNA samples
are damaged by exposure to UV light. It has recently been documented by
Epicentre Technologies that a 10-20 second exposure to 305 nm UV light on
a transilluminator is sufficient to cause extensive damage to the DNA.
This period of time is the absolute minimum required to excise a DNA band
from a gel.
An alternative to UV transillumination involves the use of laser light
sources. However, the use of laser light is not applicable to the simple
and direct viewing of a DNA gel by the human eye. The extremely small
cross-section of the laser light beam requires that a typical DNA gel be
scanned by the laser, the fluorescence intensity at each point measured
electronically and stored digitally before a composite picture of the DNA
gel is assembled for viewing using computer software.
Visible light boxes for artists' uses are known to the art for visualizing
non-fluorescing materials, e.g., as described in U.S. Pat. No. 3,802,102.
The use of visible light to detect certain fluorescent dyes is suggested,
e.g., in Lightools Research web page. However, no enabling disclosure for
making such devices is provided. None of these references provides devices
or systems for viewing fluorescence patterns using visible light.
Despite the recent development of dyes fluorescing in the visible spectrum
(Haugland, R. [1996] "Handbook of Fluorescent Probes and Research
Chemicals, Sixth Edition," Molecular Probes, Inc., Eugene, OR, pp. 13-18,
25-29, 29-35), transilluminators and other devices to take advantage of
the properties of such dyes have not been made available to the public. It
is an object of this invention to provide devices and methods for directly
and indirectly viewing and measuring patterns of fluorescence not
involving the use of UV transillumination but rather being capable of
using sources of visible light such as ordinary lamps, as opposed to
lasers and the focused lights used in standard fluorometers.
All publications referred to herein are incorporated by reference.
SUMMARY
Avisible light system is provided for detection of patterns of fluorescence
emitted by fluorophors capable of emitting light of an emitted wavelength
range (emission spectrum) when excited by light of an excitation
wavelength range (excitation spectrum). In one embodiment, the excitation
wavelength range must be different from the emitted wavelength range,
although these ranges may overlap, and at least a portion of the
non-overlapping portion of the emitted wavelength range must be within the
visible spectrum. Both the exciting and emitted wavelength ranges are
within the visible spectrum.
In preferred embodiments, using color filters, light of the "excitation
type" for the fluorophor is light within the excitation wavelength range
for the fluorophor, and light of the "emitted type" is light within the
emitted wavelength range for the fluorophor. The first filter preferably
transmits at least about 70% of the light from the light source in the
excitation wavelength range, and the second filter transmits at least
about 95% of the light in the emitted wavelength range. The term "filter"
as used herein includes combinations of filters.
In other embodiments using polarizing filters, the first filter transmits
the light from the source in a narrow range of orientations, and the
second filter is oriented to exclude light from the source, i.e.,
transmits only light orthogonal to that passed by the first filter, so
that only light emitted by the fluorophor passes through the second
filter.
This invention comprises a visible light system comprising:
a) a light source capable of producing visible light of the excitation type
for the fluorophors;
b) a first optical filter placed between said light source and said
fluorophors, which is capable of transmitting light from said light source
of the excitation type for said fluorophors and of preventing transmission
of at least a portion of the light from said light source of said emitted
type; and
c) a second optical filter placed between said fluorophors and a light
detector which second filter is capable of transmitting light of said
emitted type and of preventing transmission of light from said light
source of said excitation type, to form a viewable image of the pattern of
fluorophors.
The fluorophors may be any fluorophors known or readily available to those
skilled in the art, and are preferably used in the form of fluorophors
bound to or in a biological sample. Fluorophors may be used to detect and
quantify any desired substance to which they can be attached or into which
they can be incorporated, e.g. organic molecules such as proteins, nucleic
acids, carbohydrates, pigments, and dyes, inorganic molecules such as
minerals, bacteria, eukaryotic cells, tissues and organisms. Fluorophors
may also be an intrinsic part of an organism or substance to be detected,
e.g., various dyes and pigments found in, for example, fungi, fish,
bacteria and minerals.
The system of this invention may be incorporated into an integrated device
such as a horizontal or vertical gel electrophoresis unit, scanner or
other device in which detection of fluorescence is required.
The devices and methods of this invention are especially useful for viewing
patterns, i.e., two-dimensional and three-dimensional spatial arrangements
of fluorophors. Fluorescence detectors such as found in fluorometers are
able to detect only the presence and intensity of fluorescence, and rather
than generating an image generate a stream of data which must be
interpreted by machine. The present invention allows direct viewing of
two-dimensional (or three-dimensional) patterns of fluorophors by the
human eye. Such patterns of fluorophors include the spatial arrangement of
fluorophors on DNA on a gel, or of fluorophors on a TLC plate, the spatial
distribution of fluorophors in test tubes in a rack, the spatial
distribution of fluorophors in fungus or bacteria on skin, or on meat
meant for human consumption, or the spatial arrangement of fluorescent
fish in a tank. The images of patterns of fluorescence generated by the
methods and devices of this invention may be viewed over time and may be
photographed, digitized, stored and otherwise manipulated by machine but,
in all cases, a two- or three-dimensional image is generated. The light
source should not be a laser, and any mechanical detector used herein,
like the human eye, preferably includes an array of photodetectors.
The light source should produce minimal light in the ultraviolet range,
i.e., less than 1% of its light should be in the ultraviolet range, or the
first filter should effectively screen out ultraviolet light, preferably
to a level less than 1%, to prevent damage to DNA being viewed in the
system. Even when using polarizing filters, a blue filter is preferably
used as part of or in addition to the first filter to prevent DNA damage.
Alternatively, the diffuser may be used to filter out residual UV light,
and the diffuser and first filter can be combined into one sheet of
material. (Most blue filters filter out ultraviolet light as well as
visible light in wavelengths longer than blue.)
The light detector or "viewer" used to detect the fluorescence of the
fluorophor using this system may be a viewer's eye, or a device such as an
optical scanner or charge coupled device camera for inputting a digitized
image into a computer, or a camera. Such devices may also comprise means
for quantifying the light within the emitted wavelength range reaching the
viewer, and may also comprise means, such as a properly programmed
computer as is known to the art, for converting such quantitative
measurements to values for the amount of biological material present in
the sample being measured.
The first filter is capable of filtering out light from the light source of
the emitted type for the fluorophors. This means that at least some of the
light from the light source of the emitted type is filtered out by the
first filter. In many cases, the excitation and emission spectra for the
fluorophors being used overlap. The first filter need only absorb light in
a portion of the emission spectrum, usually the upper wavelength end
thereof.
In some embodiments, the first filter may be an integral part of the
support for the fluorophor or of the material or medium containing the
fluorophor. For example, the first filter may serve as the gel support of
a transilluminator device on which fluorophor-containing material in gel
is placed. The gel itself, e.g., impregnated with pigment such as blue
pigment, may serve as the first filter.
In some embodiments, as more fully described below, the second filter may
be adapted to be placed over the human eye, e.g. as lenses for glasses to
be worn by a human viewer, or may be adapted to be attached to the lens of
an optical scanner or camera. The second filter may also serve as a safety
lid for an electrophoresis unit or as a wall for the container for the
fluorophor-containing material. The term "attached" in this context means
both removably attached or built in as an integral part of a device. Also
in some embodiments described below, the light source may be a handheld
light source held behind the sample or preferably in front of the sample
and at an angle to the viewer. The handheld unit for holding the light
source also preferably comprises the first optical filter as part of the
casing.
The fluorophor-containing material may be transparent or opaque, and the
system may be configured to allow light from the light source to pass
directly through the first filter, the fluorophor-containing material, and
the second filter to reach the viewer in the case of a transparent medium,
or to allow light from the light source to pass through the first filter
to strike the fluorophor-containing material, allowing emitted light to
"bounce" back from the medium toward the viewer, first passing through the
second filter. The configuration of optical components may occupy any
angle from just over 0.degree. to 180.degree.. The angle is that formed by
lines drawn from the lamp to the sample and from the sample to the
detector.
The term "transilluminator" as used herein means a device (other than a
fluorometer requiring placement of fluorophor-labeled sample in a
specially constructed sample holder) which allows light to shine through a
surface in or on which a fluorophor- containing material has been placed,
and includes horizontal electrophoresis devices and other devices in which
fluorescent-containing materials are distributed on a surface.
Also provided are methods for making such systems and devices incorporating
the light source and filters described above for viewing patterns of
fluorescences emitted by fluorophors, said method comprising:
(a) providing a light source capable of producing light in the visible
spectrum;
(b) placing said fluorophors spaced apart from said light source;
(c) placing a first optical filter between said light source and said
fluorophors, said filter being capable of transmitting light from said
light source of the excitation type for said fluorophors and of preventing
transmission of light from said light source of the emitted type for said
fluorophors; and
(d) placing a second optical filter between said fluorophors and a light
detector, said second filter being capable of transmitting light of said
emitted type and of preventing transmission of light from said light
source of said excitation type.
Also provided are methods for viewing a pattern of fluorescence emitted by
fluorophors capable of emitting light of an emitted type when excited by
light of an excitation type different from said emitted type, at least a
detectable portion of said emitted type being present in visible light,
said method comprising:
(a) shining visible light on said fluorophors through a first optical
filter which is capable of transmitting light of said excitation type and
of preventing transmission of light of said emitted type, whereby said
fluorophor emits light of said emitted type;
(b) passing light emitted by said fluorophor through a second optical
filter which is capable of transmitting light of said emitted type and of
absorbing light from the light source of said excitation type to form an
image of said pattern of fluorescence; and
(c) viewing said image.
Devices of this invention use visible rather than ultraviolet light for
exciting and viewing fluorescence. Preferred embodiments of this invention
using light sources of around 9 W emit even less dangerous UV light than
standard fluorescent tubes used in most offices and laboratories. Using
visible light allows the integrity of DNA being viewed to be maintained.
The devices of this invention allow detection of as little as 0.1-1 ng of
DNA, equal to or slightly better than a 312 nm UV transilluminator. Using
a charge-coupled device (CCD) camera, it is possible to detect levels as
low as tens of picograms of SYBR Gold-stained DNA. Viewing may be done by
eye or by an imaging device such as a camera or computer scanner using
both conventional photography and digital imaging systems.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a scheme illustrating the operational principles of a device of
this invention.
FIG. 2 is a graph showing the fluorescence excitation and emission spectra
of a double-stranded DNA-bound SYBR Green I nucleic acid gel stain.
FIG. 3 shows the absorbance spectrum of the Acrylite #668-0GP optical
filter used as a first optical filter in a preferred embodiment of this
invention.
FIG. 4 shows the absorbance spectrum of the Wratten #98 optical filter used
as a first optical filter in a preferred embodiment of this invention.
FIG. 5 shows the absorbance spectrum of the Perspex #300 optical filter
used as a second optical filter in a preferred embodiment of this
invention.
FIG. 6 shows the absorbance spectrum of the Wratten #12 optical filter used
as a second optical filter in a preferred embodiment of this invention.
FIG. 7 shows absorbance spectra of the Acrylite #668-0GP and Perspex #300
optical filters used as a combination of first and second filters in a
preferred embodiment of this invention.
FIG. 8 shows absorbance spectra of the Wratten #98 and Wratten #12 optical
filters used as a combination of first and second filters in a preferred
embodiment of this invention.
FIG. 9 is a cutaway and exploded view of a transilluminator device of this
invention.
FIG. 10 is a cutaway view of an integrated transilluminator and horizontal
electrophoresis unit of this invention.
FIG. 11 is a side view of an integrated scanner-transilluminator device of
this invention.
FIG. 12 is a perspective view of a handheld unit of this invention.
FIG. 13 shows a scheme for a transilluminatorfor viewing fluorescent
materials in gels and other transparent media.
FIG. 14 shows a scheme for a top-illuminator for viewing fluorescent
materials in opaque media such as thin-layer chromatography plates.
FIG. 15 shows a scheme for viewing the position of fluorescent materials
during column chromatography.
FIG. 16 shows a gel electrophoresis apparatus in which the two plates
containing the gel also act as the two filters, allowing fluorescent
materials to be viewed during electrophoresis.
FIG. 17 shows a thin-layer chromatography apparatus in which the filters
are an integral part of the apparatus, allowing fluorescent materials to
be viewed during thin-layer chromatography.
FIG. 18 shows a handheld unit in combination with glasses worn by the
viewer having as lenses the second optical filter.
FIG. 19 shows a transilluminator of this invention comprising a handheld
second filter useful to manually scan fluorescent materials and quantitate
amounts present.
FIG. 20 compares SYBR Gold-stained DNA gels on a 312 nm UV transilluminator
and on a transilluminator of this invention.
FIG. 21 shows a SYBR Gold-stained DNA gel image captured by computer
scanning.
FIG. 22 shows gels comparing DNA degradation using a 312 nm UV
transilluminator with DNA degradation using a transilluminator of this
invention.
FIG. 23 compares ethidium bromide-stained DNA gels on an ultraviolet
transilluminator (left side) and on a transilluminator of this invention
(right side).
DETAILED DESCRIPTION
What the human eye perceives as "white light" consists of all the
electromagnetic radiation with wavelengths between approximately 400 and
750 nm (the "visible spectrum"). (Light from 200-400 nm is called
ultraviolet or UV.) Different wavelengths of light, when isolated, are
seen by the human eye as being colored: light of wavelengths between
400-500 nm is generally seen as violetblue hues; 500-550 nm is seen as
green/yellow hues; and 550-750 nm is seen as orange/red hues. The term
"visible light" as used herein refers to light having wavelength(s)
between about 400 nm and about 750 nm. Not all wavelengths in this range
need to be present in the "visible light" for purposes of this invention.
Many dyes are excited to fluoresce by light within the visible spectrum.
However, prior to the present invention, this fluorescence has not been
used in transilluminators or in handlamps because when white or broad-band
visible light is used for excitation of the dye, the fluorescence is not
detectable due to the large amount of incident light from the light source
itself that reaches the observer or detecting instrument. This problem is
overcome in the present invention by placing suitable optical filters on
either side of the material to which the fluorophor is bound to prevent
the totality of the lamp light from reaching the observer and allow the
fluorescent light from the fluorophor to be seen.
"Optical filters" remove or "absorb," i.e., prevent transmission of, light
of a certain type while allowing the passage or "transmittance" of light
of another type. For example, a color filter that appears blue is
absorbing most of the green and red light and transmitting the blue light.
A color filter that appears amber is absorbing blue light and transmitting
green and red light. The combination of green and red light appears
yellow-orange to the eye, giving the filter a yellow-orange or amber
color.
The exact optical properties of a color filter are due to the light
absorption properties of the particular pigments embedded in its matrix.
The filter matrix itself may be made from a wide range of materials known
to the art and available to the skilled worker including plastics, such as
acrylics, gelatin and glass.
Another type of optical filter is a polarizing filter. A polarizing filter
transmits light of only a narrow range of orientations and prevents
transmission of light of other orientations.
The optical properties of filters are measured in terms of either the
"absorbance" or "percent transmittance." The terms are related as shown
below:
A=-log(%T/100)
where A is the absorbance of the filter and %T is the percent
transmittance.
"Fluorescence" is the phenomenon in which light energy ("exciting light")
is absorbed by a molecule resulting in the molecule becoming "excited."
(Lakowicz, J. R. (1983) "Principles of Fluorescence Spectroscopy," Plenum
Press, New York.) After a very brief interval, the absorbed light energy
is emitted by the excited molecule, usually at a longer wavelength than
the exciting light. This emitted light is referred to as fluorescent
light. A molecule that exhibits fluorescence is referred to as a
"fluorophor." Any given fluorophor will be excited to fluoresce more by
some wavelengths of lightthan otherwavelengths. The relationship between
wavelengths of light and degree of excitation of a given fluorophor at
that wavelength is described by the "excitation spectrum" of the
fluorophor. The excitation spectrum is also called the "excitation
wavelength range" herein.
Likewise, any given fluorophor will produce more intense fluorescence at
particular wavelengths than others. The exact relationship between the
wavelength of light and the intensity of the fluorescence emission at that
wavelength is described by the "emission spectrum" or "fluorescence
spectrum" of the fluorophor. The emission spectrum is also called the
"emitted wavelength range" herein. FIG. 2 graphs the fluorescence
excitation and emission spectra of a double-stranded DNA-bound SYBR Green
I nucleic acid gel stain as taken from R. Haugland (1996) "Handbook of
Fluorescent Probes and Research Chemicals."
The excitation maximum is the wavelength of exciting light at which
fluorescence of the fluorophor reaches maximum intensity. The emission
maximum is the wavelength of light emitted by the excited fluorophor when
its fluorescence is at maximum intensity.
Most fluorophors excited by and emitting visible light have an emission
spectrum overlapping their excitation spectrum, although the maximum for
each is different. The distance in nanometers between the excitation
spectrum maximum and the emission spectrum maximum is known as the
"Stokes' shift." Fluorophors with large Stokes' shifts in the visible
range work best in this invention. For example, a fluorophor with an
excitation maximum of 450 nm and an emission maximum of 600 nm with no
overlapping between the spectra would be ideal; however most fluorophors
have smaller Stokes' shifts. For example, SYPR.RTM. Orange has a Stokes'
shift of 105 nm and SYPRO Gold has a Stokes' shift of 42 nm, while
fluorescein has a Stokes' shift of 25 nm.
Visible light sources of this invention typically emit light which includes
or overlaps both spectra. Most color filters do not sharply transmit light
only within a certain wavelength, and sharply prevent transmission of all
light outside this wavelength. Instead, as shown in FIGS. 3 and 4, most
filters allow passage of a small quantity of light even at wavelengths
where they are most effective as filters, and they prevent transmittance
of a small quantity of light at wavelengths where they are least effective
as filters for absorbing light. In a "crossover" wavelength range, the
capability of such color filters to absorb light changes (gradually or
sharply) along the wavelength scale from a region where maximum light is
being absorbed, known as the "cut-off region," to a region where most of
the light is being transmitted and only a small amount is being absorbed.
As a practical matter, the light source will produce light in wavelengths
overlapping those emitted by the fluorophor, and the filter between the
fluorophor and the viewer used to transmit light in the emission spectrum
will also allow enough light from the source to pass through to overwhelm
the fluorescence (emitted spectrum). Thus a filter placed between the
light source and the fluorophor to remove light from the source not
removed by the filter between the fluorophor and the viewer must be used.
To optimize the sensitivity of the system, filter pairs should be chosen
so as to allow viewing of (a) the maximum fluorescence intensity and (b)
minimum lamp light intensity. For typical fluorophors this involves a
tradeoff between (a) and (b). The system can be adjusted to minimize lamp
light intensity so that the lamp light does not overpower the
fluorescence.
A first consideration is to choose the filter pairs so that in combination
they prevent transmission of essentially all the exciting light to the
viewer. To achieve this, assuming the lamp produces light as close to the
excitation maximum of the fluorophor as possible: (a) the first filter
must absorb as much light as possible in the emission spectrum of the
fluorophor, i.e., in general the cut-off region must extend as far into
the blue (shorter wavelengths) as practicable; and (b) the second filter
must absorb as much light as possible in the excitation spectrum of the
fluorophor, i.e., the cut-off region must extend as far into the red
(longer wavelengths) as practicable. This tends to result in the use of
filters whose crossover regions are far apart and not overlapping.
A second consideration for choosing the filter pairs is to maximize the
amount of light in the emission spectrum for the fluorophor that reaches
the viewer: (a) the first filter should be selected to transmit as much
light as possible in the region of excitation maximum of the fluorophor;
and (b) the second filter should be selected to transmit as much light as
possible in the region of the emission maximum of the fluorophor. This
tends to result in the use of filters whose crossover regions overlap. The
point along the wavelength range where the absorbances of the two filters
coincide should be at as high an absorbency as practicable.
If the maximum ofthe emission spectrum for a fluorophor is greater than 500
nm, the absorbance of the filter selected to be placed between the
fluorophor and the light source may rise to near 4 from less than 1 in a
crossover region, e.g., from about 450 nm to about 500 nm. (Good filters
have a crossover region of less than about 50 nm.) The absorbance of the
second filter between the fluorophor and the viewer should then drop from
near 4 to less than 1 in the same crossover region such that the sum of
the absorbances of the filters at wavelengths in the crossover region is
near 4 to filter out most of the wavelengths in this region so that light
below about 525 nm is effectively prevented from reaching the viewer. Thus
the viewer sees substantially only light emitted by the fluorophor.
As discussed above, the excitation and emitted wavelength ranges of the
fluorophor can overlap. The only requirement is that light of sufficient
intensity to be detectable in a darkened space (preferably by the viewer's
unaided eye but alternatively by an optical instrument such as a camera or
optical scanner) be emitted by the fluorophor outside the excitation
wavelength range so that it can be detected after light in the excitation
wavelength range has been filtered out.
Typical fluorophors include many organic dyes. However, most molecules of
biological origin such as nucleic acids, proteins, lipids and coenzymes
are not strongly fluorescent. (Notable exceptions include Green
Fluorescent Protein and its derivatives and various pigments such as
chlorophyll and others used for coloration of plants and animals.)
Therefore, to detect biological molecules it is usually necessary to
either stain or react a biological sample with a fluorophor. "Staining"
usually refers to the process in which a fluorescent dye binds relatively
weakly to a target molecule without the formation of covalent bonds. If a
fluorophor is "reacted" with a target molecule, this usually implies that
the complex between the two species involves a relatively robust covalent
bond.
The fluorescence intensity of a sample can be used either qualitatively to
determine the presence or location of a fluorophor or quantitatively to
determine the amount of fluorophor present. Variants on measuring the
intensity of fluorescence include fluorescence resonance energy transfer
and fluorescence polarization.
Alternatively, a fluorophor may be used indirectly to reveal the presence
of a particular species. For example, the Vistra ECF Substrate system
(Amersham Life Science Inc., Arlington Heights, Ill.) involves the use of
the enzyme alkaline phosphatase, conjugated to an antibody that can bind
specially prepared DNA oligonucleotide probes, to generate a fluorescent
species. The enzymatic reaction generates multiple fluorophors,
effectively providing an "amplified" fluorescence signal from the target
DNA. Some examples of fluorophors used with biological samples are given
in Table 1.
TABLE 1
Excitation Emission
Maximum Maximum
Dye (nm) (nm Uses
ethidium bromide (EB).sup.1 518 605 stain for nucleic
acids
SYBR .RTM. Green.sup.2 494 521 stain for nucleic
acids
SYPRO .RTM. Orange.sup.5 485 590 stain for proteins
SYBR .RTM. Gold.sup.2 495 537 stain for nucleic
acids
GelStar .RTM..sup.3 493 527 stain for nucleic
acids
Vistra .TM. Green.sup.4 497 520 stain for nucleic
acids
Vistra .TM. ECF Substrate.sup.4 440 560 indirect detection
4-chloro-7-nitrobenz-2- 467 539 covalent label
oxa-1,3-diazol.sup.1
fluorescein derivatives.sup.1 495 520 covalent label
Texas Red .RTM..sup.2 587 602 covalent label
.sup.1 Available from Sigma Chemical Co., St. Louis, MO.
.sup.2 Trademark of Molecular Probes, Inc. of Eugene, OR.
.sup.3 Available from FMC Bioproducts, Rockland, ME.
.sup.4 Available from Amerisham Life Science Inc., Arlington Heights, IL.
.sup.5 SYPRO .RTM. is a trademark of Molecular Probes, Inc. of Eugene, OR.
The removal of lamp light by filters so that the viewer sees substantially
only the light emitted by the fluorophor is accomplished in two steps (see
FIG. 1). In a preferred embodiment, a filter pair comprising a blue first
filter and an amber second filter is used with a fluorophor such as
SYBR.RTM. Green I or ethidium bromide that is maximally excited at around
500 nm or less (i.e., by blue light) and emits its maximum fluorescence at
500 nm or more (i.e., the fluorescence is green or red).
The first filter, which is blue, is placed between the light source and the
fluorophor and absorbs the green and red components of the visible light
and transmits only blue light through to the fluorophor. The blue light
excites the fluorophor to fluoresce. Between the fluorophor and observer
is placed a second filter, which is amber, that absorbs the blue light
from the lamp but transmits the green or red fluorescent light from the
fluorophor to the light detector, e.g., a human viewer or detection
equipment.
Another embodiment uses polarizing filters. For a typical light source the
light is polarized equally around all possible orientations. By placing a
polarizing filter in front of a lamp it is possible to select light with a
narrow range of orientations. If a second polarizing filter is placed on
top of the first filter but orthogonal to the first, then this second
filter will remove essentially all of the polarized light that has passed
through the first filter. The net result is that no light reaches the
viewer. When a fluorescent sample is placed on top of the first filter,
the some of the sample will be excited by the polarized light that passes
through the first filter. The sample will emit fluorescence. This
fluorescence is also polarized. However, the emitted light will have a
fairly broad distribution of orientations. Some of these orientations will
be able to pass through the second filter and reach the viewer. The net
result is that the fluorescence can be seen by the viewer against a dark
background.
The "light source" used in this invention is any device capable of emitting
visible light e.g., a typical household light such as a low-powered
fluorescent tube or incandescent bulb that produces visible light
including wavelengths within the excitation spectrum of the fluorophor.
Different lamps produce different intensities of light at different
wavelengths. Thus, for example, by altering the phosphor in a fluorescent
tube, a lamp that will have maximum light output at wavelengths where
excitation of the fluorophor is maximal may be manufactured. Some examples
are given in Table 2.
TABLE 2
Maximum Output Half width of Relative Output at
Lamp (nm) Output (nm) Maximum
Phillips F40B.sup.1 460 160 0.19
Interelectric 445 33 1.00
F40T12/BBY.sup.2
Nichia NP-160.sup.3 480 120 0.35
Panasonic
FPL28EB.sup.4 blue
Panasonic
FML27EB.sup.4 blue
Sylvania 457 46
CF9DS/blue.sup.5
Dulux S9W 550* 25
F9TT/Green.sup.6
Dulux S9W
F9TT/Red.sup.6 red
.sup.1 Available from Phillips Lighting Co. of Somerset, NJ.
.sup.2 Available from Interelectric Corporation, Warren, NJ.
.sup.3 Available from Nichia America Corporation of Mountville, PA.
.sup.4 Available from Matsushita Home and Commercial Products Company,
Secaucus, NJ.
.sup.5 Available from Osram Sylvania, Inc., Maybrook, NY.
.sup.6 Available from Osram Corporation, Montgomery, NY
*Main peak.
The first optical filter is placed between the fluorophor and the light
source and transmits light from the light source in the wavelength range
of the excitation spectrum of the fluorophor. As most fluorophors useful
with the invention are maximally excited between about 450 nm and 550 nm,
the first optical filter will typically appear blue or 30 green to the
eye.
It is essential the first optical filter also prevent the transmittance of
(absorb) as much light as possible from the light source that is of
similar wavelengths to the fluorescence emission of the fluorophor. A
filter with a percent transmittance of around 0.01% at wavelengths in the
emission spectrum of the fluorophor is desirable.
Examples of filters with these properties include Acrylite.RTM. #668-0GP,
available from Cyro Industries of Rockaway, NJ and Wratten #98, made by
Eastman Kodak Company of Rochester, N.Y. FIG. 3 shows the absorbance
spectrum of this Acrylite #668-0GP filter measured by an instrument
capable of measuring absorbances up to 3.5. FIG. 4 shows the absorbance
spectrum of the Wratten #98 filter from the Kodak Photographic Filters
Handbook. The data set does not extend above absorbances of 3.0.
The second optical filter should transmit only light with wavelengths in
the region of the fluorescence emission spectrum of the fluorophor. As
most fluorophors useful with the invention have emission spectra between
about 500 nm and about 650 nm, the second filter will typically appear
yellow, amber or red to the eye.
It is essential the second filter effectively prevent the transmittance of
as much light as possible from the lamp that is transmitted by the first
filter. For most fluorophors described herein, this means the second
filter must absorb blue light. A filter with a percent transmittance of
less than 0.1% in the blue region is desirable. Filters with these
properties include Perspex.RTM.#300 made by ICI Chemicals and Polymers
Limited of Darwen, Lancs., U.K. and Wratten #12 made by Eastman Kodak
Company of Rochester, N.Y. FIG. 5 shows the absorbance spectrum of the
Perspex.RTM. #300 filter measured by an instrument capable of measuring
absorbances up to 3.5. FIG. 6 shows the absorbance spectrum of the Wratten
#12 filter from the Kodak Photographic Filters Handbook. The data set does
not extend above absorbances of 3.0. The Acrylite #408-5GP filter made by
Cyro Industries of Rockaway, N.J., even though it has acceptable
transmittance properties, should not be used alone due to intrinsic
fluorescence.
In some cases, the use of two amber filters together may be desirable. For
example, the combination of Wratten #12 with Lee #15, made by Lee Filters,
Ltd. of Andover, Hampshire, U.K. can result in enhanced levels of
fluorescence detection due to a decrease of the background light
transmitted. In a somewhat different situation, the Acrylite #408-5GP
filter, which possesses intrinsic fluorescence, can be used if a Lee #21
filter is placed between the #408 filter and the specimen. This
effectively reduces the intrinsic fluorescence. Problems caused by
intrinsic fluorescence of the filter may be alleviated by moving the
filter farther away from the light source.
The transmittance properties of the two filters should cross over from high
to low transmittance in the case of the blue filter and low to high in the
case of the amber filter, as discussed above, in such a fashion that, in
combination, the two filters prevent the transmittance of lamp light to
the viewer.
Examples of useful filter combinations for this invention include Acrylite
#668-0GP with Perspex.RTM. #300 (FIG. 7) and Wratten #98 with Wratten #12
(FIG. 8).
FIG. 1 is a scheme illustrating the operational principles a devices of
this invention and is described with respect to preferred embodiments. A
light source 10 such as a fluorescent lamp, shines broad-band visible
light 20 indicated by the broad arrow onto a first optical filter 30 which
removes wavelengths which do not activate fluorescent emission of the
fluorophor contained on the fluorophor-containing material 50, which is
typically a gel containing stained biological material. In a preferred
embodiment, first filter 30 removes red and green light. After passing
through filter 30, broad-band visible light 20 becomes light almost
exclusively in the exciting wavelength range 40, in the preferred
embodiment, blue light, indicated by the long, narrow arrow, some of which
light passes through the fluorophor-containing material 50 and some of
which strikes the fluorophor thereon causing it to emit light in the
emission wavelength range which is mixed with a large excess of light in
the exciting wavelength range to form mixed light 70, in the preferred
embodiment, red or green light mixed with blue light. Mixed light 70
passes through second optical filter 60 where light in the exciting
wavelength range (blue light) is removed leaving light in the emitting
wavelength 80, in the preferred embodiment, red or green light, remaining
to strike the light detector 90 which may be a human eye or a device such
as an optical scanner or camera. In a preferred embodiment, light source
10 is contained within a light box 15 (see FIGS. 9-12), such as a
conventional, commercially available visible light transilluminator. The
light source 10 is preferably a fluorescent tube lamp or lamps of standard
design, for example FPL28EB available from Matsushita Home and Commercial
Products Company of Secaucus, N.J. or CF9DS/blue available from Osram
Sylvania, Inc., Maybrook, N.Y. The sensitivity of the device may be
enhanced by using lamps that provide the maximum light output in the
region of the exciting light spectrum (between 450 and 500 nm in the
preferred embodiment). First filter 30 is preferably a piece of
semi-transparent material attached to the top of the light box 15 of
sufficient size to cover the entire surface of the transilluminator. The
optical properties of the sheet in the preferred embodiment are such as to
allow through light of less than about 500-550 nm and cut off light of
longerwavelengths. Any type of film or screen with these optical
properties may be used. A preferred embodiment uses an Acrylite #668-0GP
filter. The fluorophor-containing material 50 is preferably a
fluorescently stained DNA gel. Second optical filter 60 may be in the form
of a sheet directly over the gel or attached to an imaging device or in
the form of lenses for glasses 28 (shown in FIG. 14). This filter 60 is a
semi-transparent film or sheet that cuts off light of wavelengths less
than the emitting wavelength range, or at least the emitting wavelength
maximum, i.e., less than about 500-550 nm in the preferred embodiment, and
allows through light of longer wavelengths. Any type of film or screen
with these optical properties may be used. A preferred embodiment uses the
Perspex.RTM.#300 filter. When the second filter 60 is a sheet, it is
placed on top of the gel in the preferred embodiment, and is supported
along the edges to avoid contact with the gel. This filter 60 may be
attached to the light box by a hinge or other device known to the art if
desired.
The light source can be of many types and incorporated into many
structures. Any suitable source of light capable of illuminating the
entire sample in the exciting wavelength range for the fluorophor being
used may be employed as light source 10, for example a TV screen,
photocopier, overhead projector, slide projector, camera flash, street
light, strobe light, car headlight, computer scanner, or light-emitting
diode may be used.
The systems of this invention may be used for both quantitative and
qualitative analysis, detection, imaging, spectroscopy, chromatography,
microscopy, DNA sequencing, cloning, polymerase chain reaction (PCR)
processes, cell sorting, repair of DNA damage or mutation, e.g. due to
aging or cancer, live animal studies, e.g., genetically altered mice
containing the gene for green fluorescent protein, and the like, bacterial
identification, detection and growth monitoring, medical diagnosis, e.g.,
detection of fungal infections on skin, industrial and environmental
studies, mineral studies, and hobbies, e.g., the enjoyment of tropical
fish and other tropical marine species that naturally contain fluorescent
pigments.
In a preferred embodiment, an agarose or polyacrylamide gel in which DNA
fragments have been previously separated by electrophoresis is stained
with a suitable fluorescent dye such as ethidium bromide as described in a
standard manual of laboratory techniques in molecular biology, or in the
case of SYBR Green I and SYBR Gold, as described in the literature
provided by Molecular Probes, Inc. of Eugene, OR.
The stained gel, referred to herein as the fluorophor-containing material
50, is placed on top of (in front of with respect to the viewer) first
optical filter 30. The lamps or light sources 10 in the transilluminator
are switched on. Either second optical filter 60 is placed over (in front
of as defined by the viewer) the fluorophor-containing material 50, i.e.,
the gel, or glasses 28 as shown in FIG. 18, are put on by the human
viewer.
Alternatively lenses designed to attach to an optical scanner or camera
used as a viewer may embody second optical filter 60.
FIG. 9 is a cutaway view of a transilluminator device of this invention.
Light box 15 contains electrical components, supports acrylic sheets and
directs light as evenly and intensely out of the top as possible. The
inside of the box is preferably made of a white plastic to reflect as much
light as possible. The box preferably has curved edges and a reflector
under the lamp to aid reflection. The sides are angled to aid light
reflection. It is also substantially watertight and light-tight. Second
optical filter 60 is preferably an amber screen comprised of
Perspex.RTM.#300 acrylic which is designed to fit snugly over the top of
the box and to drop down over the edge of first optical filter 30, which
is preferably a blue screen. The overlap of second optical filter 60 over
the edges of first optical filter 30 prevents light leakage and prevents
second optical filter 60 from slipping off. For viewing by eye, the amber
screen can be replaced by a pair of glasses with amber lenses. For viewing
by instrument, the amber screen can be replaced by a small filter over the
viewing instrument aperture. Other materials useful for the amber screen
include 0.76 cm (0.3 in.) VSA orange vinyl from Northwest Laminating
Company, Inc., of Seattle, Wash. and Wratten filter #21 from Eastman Kodak
Co., --Rochester, N.Y. The blue screen is preferably constructed from
0.635 cm (1/4 in. ) Cyro Industries 668-0GP acrylic, Rockaway, N.J. It is
preferably attached to light box 15 in such a way that its top surface is
free of joins, holes, screws, and the like to prevent corrosion by
liquids. The screen may additionally be hardened to prevent scratching. It
may also be hinged so that the transilluminator can be used as a white
light transilluminator if desired. To be used in daylight or lit space,
the transilluminator is equipped with a viewing box, i.e., a cover over
the transilluminator through the top of which the samples can be viewed.
In a preferred embodiment, beneath the first optical filter, resting on a
lip provided by flaring the vertical sides of the light box, is a diffuser
screen 35 to provide as intense and even a light as possible across the
surface area of first optical filter 30. Preferably, the diffuser screen
35 is made of 0.16 cm (1/16 in.) white acrylic, such as Acrylite #020-4 of
Cyro Industries. Within light box 15 is disposed on/off switch 95, mains
cable 96, and fuse 97. The device may be designed for AC or DC current. AC
Ballast 99 is a magnetic ballast for the AC version of the lamp. Light
source 10 may be a single 9 W 16.5 cm (61/2 in.) blue compact fluorescent
lamp CF9DS/blue from Osram/Sylvania, Inc., Maybrook, N.Y., attached to a
vertical area of the back wall and centrally located to ensure even light
distribution. A larger version of the transilluminator contains two 28 W
fluorescent lamps (FDL28EB) available from Matsushita Home and Commercial
Company, Secaucus, N.J.
FIG. 10 is a cutaway view of an integrated transilluminator and horizontal
electrophoresis unit of this invention. The unit comprises female
connectors 100 from a DC power supply (not shown), designed to mate with
male connectors 110 placed behind or through second filter 60 which is
separated from the main portion of light box 15 by blocks 105. The DC
power supply via platinum electrode 200 supplies voltage across a gel to
fractionate a DNA sample. The second filter also serves as a safety lid.
First filter 30 also serves as a bed for the agarose gel which acts as
fluorophor-containing material 50. Dam support strips 120 and dam support
panel 125 support dam spring 122 which is made of spring-loaded plastic
and squeezed to fit between dam support 120 and first filter support 32.
Teflon-coated foam 124 is attached to dam spring 122 so that it is forced
against the gel support 32 to form a water-tight seal. A similar dam (not
shown) is placed on the left side of the device. The dams are used to
contain the liquid agarose as it gels. Comb 115 functions to provide wells
in the agarose gel into which samples may be loaded. Diffuser 35 is
disposed between first filter 30 and light source 10 to spread the light
evenly. Reservoirs 130 hold buffer. AC Ballast 99 for the light source is
disposed beneath one of the reservoirs 130, connectable to an AC power
supply via mains cable 96. Alternatively, the light source may be powered
from a DC source.
In operation, a DNA sample is incubated with SYBR Green I diluted 100- or
1000-fold in TAE, loading buffer is added and then the sample is loaded
into a well in the agarose gel. The sample is then electrophoresed at
around 100 V 50 mA. The light source is switched on. DNA fragments are
viewed as they separate. Once a DNA band of interest is separated from the
rest of the mixture, the electrophoresis can be stopped and the gel
photographed and the band cutout if desired. For simple mixtures,
different DNA bands become separated in minutes. Thus the device
dramatically reduces standard "blind" UV electrophoresis time of about two
hours. DNA samples can also be prestained, such as with ethidium bromide,
and viewed as they fractionate.
FIG. 11 shows a side view of an integrated scanner-transilluminator device
of this invention using a modified commercially available scanner. Light
sources 10 are contained within lid 140, as is first filter 30. This lid
may be used to replace the standard transparency attachment on many
scanners. Lid 140 is preferably rotatably connected, e.g. by means of
hinges (not shown) to the photodetector container 190, the top surface of
which comprises second filter 60 designed so the gel is not squashed when
the lid 140 is lowered. Photodetectors 150 disposed within container 190
move on a track 160 or are moved by other means known to the art to scan a
fluorophor-containing material 50 placed atop second filter 60.
Photodetector container 190 also comprises means for detecting the
fluorescent light, digitizing the scanned image (not shown) such as a
processor comprising scanner software (not shown) known to the art, and
digitalized image data 170 is sent to a computer (not shown) for analysis.
Sensitivity of most commercially available scanners should be improved
about 40-fold, e.g. by slowing the scan speed of the photodetectors 150,
or by replacing the photo diode array with more sensitive means such as a
charge-coupled device, for use in this invention.
FIG. 12 is a perspective view of a handheld unit 25 of this invention
designed for compactness so that the unit can be easily handheld.
Preferably, the unit uses replaceable components, and in a preferred
embodiment has dimensions of approximately (L.times.W.times.H):
27.9.times.6.35.times.3.8 cm (11.times.2.5.times.1.5 in). The unit
comprises upper casing 175, containing first filter 30 and diffuser (not
separately shown), on/off switch 95, DC ballast 98 and DC input socket
215. The light source 10 is removably mounted in lamp ballast mounting
panel 180 in lower casing 185 designed to fit and be held by screws or
latching means (not shown) into upper casing 175 so that first filter 30
is positioned directly over light source 10 when the unit is assembled.
The unit also includes a DC input jack holder 220 to allow connection to a
plug-in wall transformer to transform AC to DC.
The devices of this invention may be powered by AC or DC power using either
a magnetic, electronic or DC ballast to drive the light sources. A 12-Volt
DC power supply is preferred, as 12 V is significantly safer than 120 V.
By connecting the unit to an AC power source through a plug-in wall
transformer or the like capable of converting AC to DC, the unit can be
made adaptable to differing types of AC power available anywhere in the
world. Consequently, each assembled unit is internally identical. In
addition, the unit may be powered by rechargeable batteries. Such a
feature is particularly useful for a hand lamp, e.g., for use in hospitals
and investigations of environmental features, e.g., at crime scenes and on
or from other planets.
For increased sensitivity, lamps backed by reflective silver metallic
linings to reflect light may be used. Lamps using different phosphors and
shapes, and different wavelengths to optimize viewing of fluorescence may
also be used, for example custom-manufactured lamps. The first optical
filter may comprise separate regions for different viewing activities,
e.g., viewing dyes with different fluorescent properties, and the second
filter may comprise corresponding separate regions for viewing fluorescent
species and colored stains as ordinarily viewed with a standard light box.
The first filter may comprise slots or other means for assuring placement
with respect to a light box or may comprise other holders for the light
source. The first filter may also be rotatable in order to economize on
the footprint of the unit. The second filter may be attached to the light
box by a hinged top panel with slots for different filters if desired.
SYBR Green and SYBR Gold of Molecular Probes, Inc., of Eugene Oreg. are
preferred stains for DNA. They are more sensitive for detection of DNA
than ethidium bromide and less mutagenic. In addition, if SYBR Green is
used as a pre-stain, the cost per gel is comparable to that of using
ethidium bromide. This stain does not interfere with post-gel
manipulations of stained DNA, and if necessary, can be removed by ethanol
precipitation.
A preferred embodiment of the transilluminator of this invention comprises
a 14.times.21 cm viewing surface convenient for viewing smaller size gels.
Larger viewing surfaces, such as 28.times.42 cm may be used for multiple
and extra large gels. It is economically feasible using this invention to
make transilluminators that are far larger than known UV boxes, i.e., over
four feet long.
An optimum configuration of the device can be defined as the configuration
of lamp and filters that results, for any given fluorophor, in the maximum
amount of fluorescence and the minimum amount of lamp light reaching the
human viewer or detector.
The process of optimization begins with a consideration of the optical
properties of the particular fluorophor to be detected:
(a) The lamp should produce its maximum light intensity at wavelengths
within the excitation spectrum of the fluorophor.
(b) The first filter should transmit the maximum amount of light at
wavelengths within the excitation spectrum of the fluorophor. Filters of
the preferred embodiments hereof transmit over 70% light in this region.
(c) The second filter should transmit the maximum amount of light at
wavelengths within the emission spectrum of the fluorophor. In practice,
filters of the preferred embodiments hereof transmit over 95% of the light
in this region.
At the same time that excitation light to, and emitted light from, the
fluorophor are maximized, it is essential to keep the light from the lamp
that reaches the viewer to a minimum. This involves the following
considerations:
(a) A lamp that produces minimal light intensity outside the excitation
region of the fluorophor.
(b) The first filter should absorb as much as possible of the lamp light
with wavelengths outside the excitation spectrum of the fluorophor.
Filters of the preferred embodiments hereof absorb about 99.99% of the
light in this region.
(c) The second filter should absorb as much as possible of the lamp light
with wavelengths outside of the emission spectrum of the fluorophor.
Filters of the preferred embodiments hereof absorb about 99.9% of the
light in this region.
(d) The absorbing wavelength regions of the two filters must cross over
such that the sum of the absorbances of the two filters in the crossover
region results in as much as possible of the lamp light in this region
being absorbed. In practice, the best filter combinations found so far
absorb about 99.9% of the light in this region.
(e) If the first filter transmits lamp light in a region outside the
excitation or emission regions of the fluorophor, then the second filter
must absorb this light.
(f) If the second filter possesses intrinsic fluorescence, it should also
comprise an auxiliary second filter placed between it and the light source
to filter out light which excites it to fluoresce.
In optimizing the system for the detection of a particular fluorophor, a
lamp containing a specially designed phosphor may be used, or filters
containing specially designed pigments may be used, as may be readily made
and assembled by one skilled in the art without undue experimentation.
Using readily available components, the following optimal configuration has
been established for a light box to detect DNA fragments separated by gel
electrophoresis and subsequently stained with SYBR Green I or ethidium
bromide:
(a) lamp: Panasonic FPL28EB (available from Matsushita Home and Commercial
Products Company, Secaucus, N.J.) or Sylvania CF 9 DS/blue
(b) first filter: Acrylite #668-0GP
(c) second filter: Perspex.RTM.#300
With these components it is possible to construct a transilluminator that
provides a comparable level of sensitivity for the detection of stained
DNA to that of a 14,--, conventional UV transilluminator, as described in
the Example below (see Table 7).
This configuration of lamp and filters is also appropriate for detecting
other fluorophors with similar excitation and emission properties to SYBR
Green I and ethidium bromide, such as SYPRO Orange, Vistra Green, Vistra
ECF substrate, GelStar, fluorescein and derivatives, and eosin and
derivatives, and rhodamine and derivatives.
The principles described herein can be used to make a large number of
different devices using various arrangements of the components.
For example, FIG. 13 shows a scheme for a transilluminator for viewing
fluorescent materials in gels and other transparent media. In this
embodiment, light sources 10 and first optical filter 30 are contained in
a holder or light box 15, atop which the fluorophor-containing material 50
is placed. Second optical filter 60 is placed over the
fluorophor-containing material 50. Light in the exciting wavelength range
hits first filter 30 to filter out other wavelengths, and passes into
medium 50 causing fluorophors therein to fluoresce, emitting light in the
emitted wavelength range which, mixed with light in the exciting
wavelength range, passes through second optical filter 60 where light in
the exciting wavelength range is filtered out, leaving substantially only
light in the emitted wavelength range to strike the light detector 90.
FIG. 14 shows a scheme for an epi-illuminator for top illumination for
viewing fluorescent materials in opaque media such as thin-layer
chromatography plates. In this instance, light from the light sources 10
held in light box 15 passes through first optical filter 30, to excite
fluorophors in medium 50 to emit light in the emitted wavelength range
which passes through second filter 60 placed at an angle (preferably, but
not necessarily 90.degree. ) to first filter 30 for filtering out
wavelengths other than those in the emitted wavelength range, after which
the light in the emitted wavelength range strikes the light detector 90.
FIG. 15 shows a scheme for viewing the position of fluorescent materials
during column chromatography. In this case, a light box 15 containing
light sources 10 and first filter 30 is placed next to the
fluorophor-containing material 50, a column chromatograph. Second filter
60 is placed on the opposite side of the column. Light passes through the
first filter 30, hits the column 50, and passes through second filter 60
to the light detector 90.
FIG. 16 shows a gel electrophoresis apparatus in which the two plates
containing the gel also act as the two filters, allowing fluorescent
materials to be viewed continuously during electrophoresis. Light box 15
containing light sources 10 holds first filter 30 in place. First filter
30 and second filter 60 act as the two plates holding the gel, i.e. the
fluorophor-containing material 50. The light detector 90 is placed so as
to receive light passing from the light sources 10 through first filter
30, the fluorophor-containing material 50 and second filter 60.
Preferably, the horizontal electrophoresis transilluminator of this
invention has a footprint of about 25.times.10 cm, and is the same size as
an ordinary gel box. Since the viewer can continuously monitor the
progress of a DNA fractionation, a gel only needs to be run until the DNA
band(s) of interest are separated, thus in many cases, gel running times
can be cut to fifteen to twenty minutes. In addition, DNA bands can be
excised out of the gel in the electrophoresis unit, avoiding the danger of
damaging the gel during transfer to a separate transilluminator.
FIG. 17 shows a thin-layer chromatography apparatus in which the filters
are an integral part of the apparatus, allowing fluorescent materials to
be viewed during thin-layer chromatography. In this case, first filter 30
is an integral part of light box 15 containing light source 10, which is
detachably connected to container 27 into which the fluorophor-containing
material 50 is placed. One side of container 27 comprises second filter
60. As in FIG. 11, light from light source 10 passes through first filter
30 to strike the fluorophor-containing material 50, and the fluorescence
passes through second filter 60 and reaches the light detector 90.
FIG. 18 shows a handheld unit in combination with glasses 28 containing the
second filter 60 worn by a human viewer. The eye of the viewer, or a
mechanical light detector 90, is covered by a lens or lenses, shown as
part of glasses 28 containing second filter 60. Light from light source 10
in handheld unit 25 passes through first filter 30 also comprised in
handheld unit 25, then passes through the fluorophor-containing material
50 and second filter 60 comprised in glasses 28 to reach the viewer's eye
or light detector 90. This embodiment is useful for a transparent medium.
In alternative embodiments involving an opaque medium, the handheld unit
25 may be placed with respect to the fluorophor-containing material 50 so
that light from light source 10 hits medium 50 and fluorescence emitted
passes through to second filter 60 and light detector 90. The light source
10 may operate on DC or AC current. As a DC unit, handheld unit 25 may be
powered by rechargeable batteries and thus run in remote locations if
desired.
The handheld unit provides versatility for viewing fluorophors in both
"open" systems such as agarose gels, nitrocellulose and polyvinyl
difluoride (PVDF) membranes and thin layer chromatography (TLC) plates, as
well as "closed" systems such as plastic and glass tubes, 96-well plates,
chromatography columns, and sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS PAGE) gels and any kind of gel during
electrophoresis. Using visible light, fluorophors can be viewed through a
wide range of transparent or semi-transparent materials such as glass,
polystyrene, polyethylene, polypropylene or acrylic. For example, in 1.5
mL polypropylene centrifuge tubes, using a handheld embodiment as
described herein, fluorescein can be detected with eight times more
sensitivity than using a UV lamp such that concentrations as low as 25
nmol/L may be detected, whereas using UV light at 360 and 312 nm, about
200 nmol/L is the lowest detectable concentration of fluorescein, and
using UV light at 254 nm, over 1000 nmol/L of fluorescein must be present
to be detected.
In "open" systems such as agarose gels, nitrocellulose membranes and TLC
plates, fluorescein has been found to be detectable at very low
concentrations. For example, on PVDF membranes, the visual detection limit
is around 12 femtomoles of fluorescein, about twice the sensitivity
achieved using UV light.
FIG. 19 shows a transilluminator of this invention comprising a light box
15 containing light sources 10 and first filter 30 atop which is placed
the fluorophor-containing material 50. Handheld wand 210 comprising second
filter 60 may be manually passed overthe fluorophor-containing material 50
and sends image data 170 to a detector (not shown). The viewer 90, shown
as a human eye wearing glasses also containing second filters 60, is able
to directly view the fluorescence to aid in directing the wand over the
fluorophor-containing material.
FIG. 20 shows a gel comparing SYBR Gold-stained DNA on a 312 nm UV
transilluminator (left panel) and a transilluminator of this invention.
Various amounts of .lambda. DNA cut with Hindlil were separated by gel
electrophoresis and the gel stained with SYBR Gold. The gels were then
photographed on a 312 nm UV transilluminator (left) or a transilluminator
of this invention (right). As can be seen, the transilluminator of this
invention provides greater sensitivity.
FIG. 21 shows the SYBR Gold gel of image shown in FIG. 20 (right side) made
using a transilluminator of this invention and captured using a computer
scanner. The original colored image was converted to grayscale and
reversed.
FIG. 22 shows gels comparing DNA degradation using a 312 nm UV
transilluminator (right side) with that using a transilluminator of this
invention. 100 ng of supercoiled (sc) plasmid pBR322 containing SYBR Green
I was placed on either an embodiment (F40T12/BBY +#668 filter) or a 312 nm
UV transilluminator (UV) for various times. The DNA was then digested with
T4 endonuclease V which excises T:T dimers. The DNA was then run on a 0.7%
agarose gel and photographed. It is clear that as little as a 5 second
exposure to UV light is sufficient to convert almost 100% of the plasmid
into the relaxed (rx) form, and after 300 seconds, the DNA is completely
fragmented. In contrast, a 300 second exposure on the embodiment of this
invention resulted in no detectable alteration to the plasmid.
FIG. 23 shows gels comparing DNA stained with ethidium bromide using a
standard UV transilluminator (left side) and a transilluminator of this
invention (right side).
As will be appreciated by those of skill in the art, the second filter
shown in any of the above-described embodiments may be provided in the
form of lenses for glasses or as attachments to mechanical light detectors
rather than as a filter sheet or plate as shown. Further the devices can
be configured with interchangeable filters or side-by-side filters to
allow different fluorophors to be detected with maximum sensitivity. Lamps
may be constructed to provide wavelengths optimized for each system, all
as may be readily understood by those of skill in the art following the
teachings provided herein.
EXAMPLES
Example 1
Sensitivity
The sensitivity of an optimized device of this invention was measured by
detection of known quantities of DNA on gels stained with SYBR Green I and
ethidium bromide, both by eye and by photography. What is seen by the eye
and what is recorded on photographic film are not necessarily one and the
same, especially when using black-and-white photography. For example,
photographic film is able to accumulate an image over many seconds and,
after processing, the image can be quantitated. On the other hand, the
interpretative skill of the human eye when directly viewing an image is
unparalleled. Though scientists use photographs of DNA gels for their
laboratory records and for detailed analysis such as calculation of the
sizes of DNA fragments, much of the analysis of a DNA band pattern on a
gel is achieved using the naked eye. Furthermore, the excision of gel
slices containing DNA is always done by eye. Therefore, it is important
that the sensitivity of any apparatus for visualization of DNA in gels be
documented and optimized for human eye and photographic detection methods
separately.
The light-box used was an Apollo 100 obtained from OfficeMax, Denver, Colo.
It came equipped with an F15T8DRWG fluorescent tube. This box was
convenient for testing 45.7 cm (18' in.) fluorescent tubes such as the
Osram F15T8D and Osram F15T8BLK. Other lamps were accommodated in
makeshift housings.
Fluorescent tubes were obtained from Environmental Lighting, Denver, Colo.
(Osram F15T8D, Osram F15T8BLK, Phillips F40B and Sylvania CF9DS/blue
lamps) and U.S. Aquarium, Denver, Colo. (Panasonic FPL28EB lamps).
The gelatin filters used were obtained from Mike's Camera, Boulder, Colo.,
or from Wasatch Photographic, Denver, Colo., and included Kodak Wratten
gelatin filters #12 (yellow), #21 (amber), #98 (blue) and #47 (blue) and
Lee gelatin filters #15 (amber) and #21 (amber).
The acrylic filters used were obtained from either SS Plastics, Englewood,
Colo., Fantastic Plastic, Englewood, Colo., or Colorado Plastic, Boulder,
Colo., and included Acrylite#408-5GP (amber), Acrylite#668-0GP (blue), RAM
#UM 2119 (amber), Dupont Lucite L #AM2422 (amber) and Dupont Lucite L
#AM2424 (blue). In addition, amber filter Perspex.RTM.#300 was obtained
from Amari Plastics, Bristol, UK. All American acrylic filters were used
in a 0.32 cm (1/8 in.) inch thick sheets except the 668-0GP blue which was
used in both 0.32 cm (1/8 in.) and 0.635 cm (1/4 in.)thicknesses. The
British materials were 3 and 6 mm.
The fluorescent dyes ethidium bromide, SYBR Green I and SYBR Gold were
obtained from Molecular Probes Inc., Eugene, Oreg. All other chemicals
were obtained from Boehringer Mannheim Corporation, Ind., Ind. or Sigma
Corporation, St. Louis, Mont.
Three samples of .lambda. DNA (1 .mu.g, 0.1 .mu.g and 0.01 .mu.g) cut with
the restriction enzyme EcoRI were electrophoresed in duplicate on a 0.7%
agarose gel in 40 mmol/L tris acetate buffer (TAE), pH 7.8; 1 mmol/L
ethylene diamine tetraacetic acid (EDTA) at 85 V for 90 minutes. The gel
was then cut in half. One half of the gel was stained in a 1:10,000
dilution of SYBR Green I in TAE for 30 minutes at room temperature, and
the other half was stained in 0.5 .mu.g/mL solution of ethidium bromide in
TAE underthe same conditions. The gels were stored at 4.degree. C.
For reference purposes, the gels were photographed on a UVP model #C-63 UV
transilluminator (302 nm illumination) (Ultraviolet Products, Inc.,
Upland, Calif.) using Polaroid 667 film. The exposure time was 0.5 seconds
and the f-stop was 5.5. A Kodak Wratten #12 filter was placed on top of
the gel. The camera was an oscilloscope camera C27 (Tektronix Inc.,
Portland, Oreg.).
In order to determine the optimal configuration of filters and lamps, a
prototype visible light transilluminator was constructed according to the
scheme illustrated in FIG. 1 and described above. The gelatin filters were
enclosed in clear, transparent acrylic sheets to protect them. All filters
were enclosed in cardboard frames to prevent light leakage around the
edges. A black-out cloth was also used to eliminate stray light from the
lamp.
A variety of lamps and filters were placed in the apparatus and the DNA
bands in the gels were visualized and photographed in a dimly lit room. No
additional filter was used with the camera.
In order to compare the new transilluminator with the conventional UV
model, from the known sizes of the fragments generated by EcoRI digestion
of the A DNA, the amount of DNA in each band on the agarose gel was
calculated. The amounts ranged from 410 ng to 0.7 ng per band. A complete
listing is given in Table 3.
To provide a standard measure of detectability, the stained gel was first
placed on a standard 302 nm UV transilluminator. The DNA bands were
visible using the naked eye down to the 0.9 ng level when stained with
SYBR Green I, and 1.4 ng when stained with ethidium bromide (Table 4). In
a photograph, the sensitivity was marginally lower: 1.4 ng for SYBR Green
I and 4.4 ng for ethidium bromide. The slightly greater visibility of the
SYBR Green-stained DNA is probably due to a lower background light level
from the gel itself. The ability to detect as little as 0.9 ng of DNA
serves as a reference point for the sensitivity of the constructed visible
light transilluminator.
The gels were then placed on the new transilluminator and various
combinations of blue filters underneath the gel and amber filters above
the gel were tried together with different lamps. Both naked-eye and
photographic film results are given in Tables 5 and 6.
Of the blue filters, #2424 transmits excessive amounts of red light and its
use was not pursued any further. The #98 transmits blue light of
significantly shorter wavelengths than either #668-0GP or #47, both of
which appear to have very similar transmission characteristics. The
shorter wavelength transmission characteristics of #98 mean that it can be
used with the yellow emission filers (e.g., #12), whereas #668-0GP and #47
are optimal with the orange emission filters.
With either #668-0GP or #47 as excitation filter, it was found in general
that the use of a single orange filter on the emission side was
insufficient, either because too much background light was transmitted to
allow detection of the fluorescent DNA, or because the filter possessed
intrinsic fluorescence which obscured the DNA fluorescence. This latter
problem was particularly noticeable with filters #408 and #2422.
The filter fluorescence could be overcome by using two emission filters
in-line.
Thus, by placing a #2119 or Lee #21 before a #408 or #2422 relative to the
lamp, it was possible to significantly reduce the fluorescence of the
second emission filter.
Filter Perspex.RTM. #300 did not possess any intrinsic fluorescence and, in
combination with #668-0GP as the excitation filter, yielded the best
overall results. The photography involved significantly different exposure
times: typically, 0.5 seconds for UV, 5 seconds for the F40B, and 15
seconds for the Fl 5T8D. Using the F15T8D, the detectability of SYBR
Green-stained DNA in photographs was approximately the same using either a
five second or a 15 second exposure time. However, using a five second
exposure, the ethidium bromide-stained DNA was essentially undetectable in
photographs.
A useful arrangement involves excitation filter#668-0GP and emission
filter#300 (Table 7). Either lamp F15T8D or F40B yields similar levels of
DNA detectability to the naked eye. For photographic purposes, the F15T8D
requires a 15 second exposure to adequately reveal EB-stained DNA whereas
the F40B requires five seconds. This difference is unlikely to be of any
practical significance. A lamp readily available in a size that fits the
light-box is preferred.
The smallest amount of DNA visible to the naked eye using an F15T8D lamp
and #668-0GP and #300 filters is 0.7 ng if stained with SYBR Green I. This
is comparable to the detection level of the UV transilluminator (0.9 ng).
With ethidium-stained DNA the white light (WL) transilluminator is
somewhat less sensitive with a 4.1 ng detection level, compared to the UV
transilluminator's ability to detect 1.4 ng.
By photography, the situation is reversed: the detection level of 0.7 ng
for SYBR Green-stained DNA using the WL transilluminator is somewhat
better than the UV transilluminator (1.5 ng). With ethidium
bromide-stained DNA, both transilluminators are of comparable sensitivity
and can detect 4.1 ng of DNA.
TABLE 3
Amounts of DNA present in the gel after electrophoresis.
Size ng of DNA per band
Band No. (base pair) 1 .mu.g load 0.1 .mu.g load 0.01 .mu.g load
1 21220 410 41 4.1
2 7420 140 14 1.4
3 + 4.sup.1 5800 + 5640 220 22 2.2
5 4800 90 9 0.9
6 3530 70 7 0.7
.sup.1 Bands #3 and #4 were not resolved on the gel.
TABLE 4
Detectable levels of DNA using a UV transilluminator
Amount of DNA detectable (ng)
Method of Detection SYBR Green I Ethidium bromide
Eye 0.9 1.4
Photo.sup.1 1.5 4.1
.sup.1 The exposure time for the photographs was 0.5 second.
TABLE 5
Direct Visual Detection of Fluorescent DNA.sup.1
Blue Filter
47 98 688-0GP
WL BL WL BL WL BL
Amber Filter SG EB SG EB SG EB SG EB SG EB SG EB
12 + 15 0.9 41
2119 + 408 0.9 9 0.9 9 0.9 22 0.9 9 0.7 4.1
2119 + 2442 0.9 9 0.7 4.1 0.9 22 0.9 9 0.7 4.1
21 + 408 0.9 22 0.7 9 0.7 9 0.7 4.1
300 0.7 9 0.7 9 0.9 22 0.7 4.1 0.7 4.1
.sup.1 This table documents the minimum amount of DNA (in nanograms)
visible on the gel using various filter combinations. WL, white
fluorescent lamp F15T8D; BL, blue fluorescent lamp F40B; SG, SYBR Green I;
EB; ethidium bromide. Blank entries were not measured.
TABLE 6
Photographic Detection of Fluorescent DNA.sup.1
Blue Filter
47 98 688-0GP
WL BL WL BL WL BL
Amber Filter SG EB SG EB SG EB SG EB SG EB SG EB
12 + 15
2119 + 408 0.7 9
2119 + 0.9 9 0.9 4.1
2422
21 +408 1.0 9 0.7 9 0.9 9
300 1.0 9 1.0 9 0.7 4.1 0.7 4.1
.sup.1 This table documents the minimum amount of DNA (in nanograms)
detectable in photographs of the gel. WL, white fluorescent lamp F15T8D;
BL, blue fluorescent lamp F40B; SG, SYBR Green I; EB, ethidium bromide.
The exposure times for the WL and BL photographs were 15 seconds and five
seconds, respectively.
TABLE 7
Detectable Levels of DNA Using the Transilluminator.sup.1
Method of Amount of DNA detectable (ng)
Detection SYBR Green I stained gel Ethidium Bromide stained gel
Eye 0.7 (0.9) 4.1 (1.4)
Photo 0.7 (1.5) 4.1 (4.1)
.sup.1 The transilluminator was equipped with an F15T8D lamp and #668-0GP
(blue) and #300 (amber) filters. For the photographic detection the
exposure time was 15 seconds. The amounts of DNA detectable using a UV
transilluminator are in parentheses. (See Table 3.)
Example 2
Blue Compact Fluorescent Lamps and SYBR Gold
Various dilutions of A DNA cutwith HindllI (Boehringer Mannheim) in 10
mmol/L Tris-Cl, 1 mmol/L EDTA were incubated at 60.degree. C. for three
minutes. The samples were placed on ice and sample loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol, 15% Ficoll type 400 in 10 mM
Tris-Cl, 1 mmol/L EDTA, pH 7.5) was added to each mix. Various amounts of
the DNA samples (from 428 ng to 0.85 ng) were loaded onto a 1% agarose gel
7.6.times.12.7 cm (3".times.5"in.) in 89 nmol/L Tris borate, pH 7.82, and
2 mmol/L EDTA (TBE). The gel was run at 80 V for two hours and then placed
in 100 mL of 1:10000 dilution (in TAE) of SYBR Gold for 30 minutes. The
gel was photographed using a Polaroid.RTM. camera (Polaroid Corporation,
Cambridge, Mass. with Polaroid 667 film on either a Fisher UV 312 nm
variable intensity transilluminator (Fischer Scientific, Pittsburgh, Pa.,
Model No. FBTTV-816), set to maximum intensity in all cases, a Wratten #12
on the camera, f-stop =5.6, exposure time=1/8 second) or an embodiment of
the present invention equipped with a CF9DS/blue lamp, a #668-0GP first
filter, and a #300 second filter (no additional filter over the camera,
f-stop=5.6, exposure time=1 second).
The photographs are shown in FIG. 20. Table 8 shows the amount of DNA in
each band.
TABLE 8
The Amount of .lambda. DNA cut with HindIII on the Gel.
The amounts of DNA listed are in ng.
Lane # 1 2 3 4 5 6 7 8 9 10
DNA 428 214 107 54 27 13 6.7 3.3 1.7 0.85
Load
Band 1 204 102 51 26 13 6.4 3.2 1.6 0.80 0.40
Band 2 83 42 21 10 5.2 2.6 1.3 0.65 0.32 0.16
Band 3 58 29 14 7.2 3.6 1.8 0.90 0.45 0.22 0.11
Band 4 38 19 10 4.8 2.4 1.2 0.60 0.30 0.15
0.075
Band 5 20 10 5.1 2.6 1.3 0.64 0.32 0.16 0.080
0.040
Band 6 18 8.9 4.5 2.2 1.1 0.56 0.27 0.13 0.070
0.035
In the photograph taken using the embodiment of this invention, it is
possible to visualize band 3 in lane 10. This corresponds to 110 pg of
DNA. In the photograph taken using the UV transilluminator it is possible
to see band 2 in lane 10. This corresponds to 160 pg of DNA.
By eye, lane 10, band 4 (75 pg) was just at the limit of visibility for
both devices.
Example 3
Ethidium Bromide Gel
DNA cut with Hindlil (Boehringer Mannheim) in 10 mmol/L Tris-C1, 1 mM EDTA
was mixed with sample loading buffer and various amounts of DNA (from 125
ng to 15.6 ng) were loaded onto a 0.7% agarose gel (3".times.5") in TAE.
Ethidium bromide was added to both the gel and running buffer to a final
concentration of 0.25 .mu.g/mL. The gel was run at 110 V for two hours and
then examined by eye and photographed using a Polaroid camera with
Polaroid 667 film on either a UV 312 nm transilluminator (Fisher
Scientific) set to maximum lamp intensity using a red Tiffen 40.5 mm 23A
filter (Tiffen Manufacturing Corp., Hauppauge, N.Y.) on the camera
(f-stop=5.6, exposure time=2 seconds) or an embodiment of the present
invention as depicted in FIG. 9 equipped with a CF9DS/blue lamp, a
#668-0GP first filter, and a Perspex.RTM. #300 second filter. A Wratten
#21 (Kodak) was used as an additional second filter for photography
(f-stop=5.6, exposure time=30 seconds). The gel was also observed and
photographed (f-stop=5.6, exposure time=10 seconds) on an embodiment
identical to the above except that it contained two CF9DS/blue lamps.
The photographs of the gel are shown in FIG. 23. Table 9 shows the amount
of DNA in each band.
TABLE 9
The amount of .lambda. DNA cut with Hind III on the Gel.
The amounts of DNA listed are in ng.
Lane # 1 2 3 4
DNA load 125 63 31 16
Band 1 60 30 15 7.5
Band 2 24 12 6.1 3.0
Band 3 17 8.5 4.2 2.1
Band 4 11 5.6 2.8 1.4
Band 5 6.0 3.0 1.5 0.75
Band 6 5.2 2.6 1.3 0.65
By eye, using the UV transilluminator, it was possible to see 0.65 ng of
DNA. Using the single lamp embodiment it was possible to see 2.4 ng of
DNA, and using the twin lamp embodiment it was possible to see 1.2 ng of
DNA. Overall, the viewability of the DNA bands in the twin lamp embodiment
was better than in the single lamp version in that the eye did not take as
long to adjust as it did to the lower light levels emanating from the
single lamp version.
In the Polaroid photograph taken using the 312 nm UV transilluminator it is
possible to see band 6 in lane 4. This corresponds to 0.65 ng of DNA. In
the photograph taken using the single lamp embodiment it is possible to
see the same band if the Polaroid film is exposed for 30 seconds. The twin
lamp embodiment gave very similar DNA detectability results in the
photograph but the exposure time required was only one third as long.
Example 4
Gel Scanning
The SYBR Gold-stained gel used in Example 2 was placed on an Astra 600S
scanner (Umax Technologies, Inc., Fremont, Calif.) linked to a Power
CenterPro.TM. 180 computer (PowerComputing, Round Rock, Tex.) running
VistaScan V2.3.7 software (Umax Data Systems, Inc.). The amber filter
Perspex #300 was placed on top of the scanner bed, the gel placed on top
of the Perspex and, on top of that, a #668-0GP first filter and a
CF9DS/blue lamp. The gel was scanned in color at 600 dpi using
"transmissive mode" with VistaScan settings of 97, 9, 34 and 53 for
highlight, shadow, brightness and contrast respectively.
FIG. 21 shows the resultant image enhanced using image manipulation
software as found in Canvas 5.0 (Deneba Systems, Inc., Miami, Fla.). It is
possible to see band I in lane 6. This corresponds to 6.4 ng of DNA.
Example 5
DNA Integrity
Supercoiled plasmid pBR322 was placed on either a transilluminator of this
invention or a 312 nm UV transilluminator for various periods of time. The
DNA was then digested with T4 endonuclease V which excises T:T dimers and
run on a 0.7% agarose gel to allow quantitation of the amount of relaxed
plasmid formed.
1 .mu.g of supercoiled pBR322 in 100 .mu.L of 50 mmol/L Tris, pH 7.5, 5
mmol/L EDTA was incubated with 1 .mu.L of a 100-fold dilution of SYBR
Green I (diluted in 50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA) on ice. This
mixture was placed directly onto the surface of either an embodiment
composed of an F40T12/BBY lamp (Interelectric Inc., Warren, N.J.) and a
Cyro #668-OGP filter, or a 312 nm UV transilluminator. A "zero-time"
aliquot of 10 .mu.L was removed from the surface before turning on the
transilluminator and stored in the dark on ice. Further 10 .mu.L samples
were removed at 5, 15, 30, 60 and 300 seconds after the device was turned
on.
1 .mu.L of a 20-fold dilution (using 50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA)
of T4 endonuclease V (Epicentre, Madison, Wis.) was added to each 10 .mu.L
time-point and allowed to react for two hours, 37.degree. C. This enzyme
excises T:T dimers. The samples were then run on a 0.7% agarose gel in TAE
and the band pattern photographed.
The results are shown in FIG. 22. UV light is shown to be extremely
damaging to DNA; after a mere five second exposure the supercoiled DNA is
almost completely converted to the relaxed form, and after five minutes
almost all the DNA has been converted into a low molecular smear. Using
the transilluminator of this invention, however, essentially no DNA damage
was detectable over the entire duration of the exposure (five minutes).
The invention maintains the integrity of the DNA samples. This feature of
the invention provides for enhanced efficiencies in procedures where the
integrity and information content of the DNA samples is important such as
gene cloning and sequencing.
Example 6
Polarization
To test the ability of a pair of polarization filters to select for
fluorescent light and to remove lamp light, an agarose gel containing
various amounts of A DNA restricted with HindIII and stained with SYBR
Gold stain (the same gel used in Example 2) was viewed using several
filter combinations. The light source was a CF9DS/blue lamp. The
polarizing filters were from Visual Pursuits, Inc., Vernon Hills, Ill.
TABLE 10
First filter Second filter ng DNA
none none 26
P* P (parallel) 83
P P (orthogonal) 5.2
*P indicates a polarization filter from Visual Pursuits.
For photography using Polaroid 667 film it was found to be necessary to
include a Wratten #21 filter to reduce the lamp light to levels at which
the fluorescent DNA bands could be captured.
The lamp light was not completely eliminated by the two orthogonal
polarizing filters, making the sensitivity of this embodiment relatively
poor. The absorption spectrum of two orthogonal polarizing filters
together revealed that a significant amount of blue light was transmitted
(%T.sub.460nm =0.23%). This indicates that these particular polarizing
filters are not polarizing the light in this wavelength range efficiently
enough to be of much practical use. Filters which, in combination, have a
%T of around 0.02% or less are required. Polarization of fluorescence may
be used to distinguish between large and small fluorophor molecules,
immobilized or free fluorophor molecules, or oriented/non-oriented
molecules.
It should be understood that the visible light fluorometric detection
system as specifically described herein could be altered without deviating
from its fundamental nature. For example, different light sources, sets
and types of filters could be substituted forthose exemplified and
described herein, so long as the light reaching the light detector, i.e.,
the viewer's eye or detection device, contains sufficient information
about the light emitted from the fluorophors whose fluorescence is being
visualized to allow viewing of an image of the pattern of fluorescence and
so long as sufficient interfering light has been filtered out such that
visualization is possible. It is therefore to be understood that within
the scope of the appended claims, the invention may be practiced in ways
other than as specifically described herein.
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